RESUMO
Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeKI [corrected], encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum pernix K1. I-ApeKI [corrected] consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5'-GCAAGGCTGAAAC downward arrowTTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3' ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites.
Assuntos
Aeropyrum/enzimologia , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Aeropyrum/genética , Sequência de Aminoácidos , Proteínas Arqueais/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Endodesoxirribonucleases/química , Íntrons , Mutação Puntual , RNA Ribossômico 16S/genética , Especificidade por SubstratoRESUMO
Homing endonucleases (HEs) of the LAGLIDADG family cleave intron/inteinless cognate DNA at, or near, the insertion site (IS) of their own intron/intein. Here, we describe a notable exception to this rule. Two introns, Pog.S1205 (length 32 bp) and Pog.S1213 (664 bp), whose ISs are 8 bp apart, exist within the 16S rRNA gene of the archaeon Pyrobaculum oguniense. Pog.S1213 harbors a nested open reading frame (ORF) encoding a 22 kDa monomeric protein, I-PogI, which contains two LAGLIDADG motifs and has optimal DNA cleavage activity at 90 degrees C. Intriguingly, I-PogI cleaves the Pog.S1205-less substrate DNA in the presence or absence of Pog.S1213. The cleavage site (CS) of I-PogI does not coincide with the IS of Pog.S1213 but with that of Pog.S1205. Thus, I-PogI activity both promotes the homing of its own intron, Pog.S1213, and guarantees co-conversion of the ORF-less intron Pog.S1205.
Assuntos
Archaea/genética , Endonucleases/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Catálise , DNA Ribossômico/metabolismo , Eletroforese em Gel de Poliacrilamida , Éxons , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Plasmídeos/metabolismo , Ligação Proteica , RNA Ribossômico 16S/metabolismo , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Some archaeal ribosomal DNA (rDNA) introns carry homing endonuclease-like genes and are therefore assumed to propagate by "intron homing". A previous study demonstrated that three introns are located within the rRNA operon (arnSL) of Aeropyrum pernix strain K1, two of which, Ialpha and Igamma, harbor open reading frames (ORFs) encoding putative LAGLIDADG-type endonucleases. In an effort to understand further the rDNA intron distribution in natural A. pernix populations, 11 A. pernix strains were isolated from marine hydrothermal biotopes, and comparative nucleotide sequence analysis of the arnSL alleles was performed. Of the 11 isolates, eight contained multiple introns, and three patterns of intron insertion were found. Three novel introns, Idelta (62 bp in length), Ivarepsilon (122 bp) and Izeta (57 bp) were identified. They were all ORF-less, but their predicted RNA secondary structure at the exon-intron junctions was consistent with the bulge-helix-bulge motif. The insertion positions and the terminal inverted repeat sequences of Idelta and Izeta were in agreement with those of Ialpha and Igamma, respectively. This suggests that these intron variants were generated by large indels (insertions/deletions) during their evolution.
